Functionalization consists of adding functional chemical groups on a surface for the purpose of modifying or improving its properties. Some of these properties can be hydrophobicity, antioxidant and/or bacteriostatic capacity, high mechanical strengths, etc. As regards the property of hydrophobicity, the absorption of liquids in the structure of the paper is a key factor for the final use of paper products (e.g. paper cups, paper bags, boxes for packaging or containers for liquids), and also for runnability of paper-making processes, e.g., in size press or printing (1, 2). Paper manufacturers perform processes to reduce the rate of liquid absorption into the structure of the paper by treating the fibrous suspension with hydrophobic substances (3); this operation is referred to as “internal sizing”. In contrast with internal sizing is “external sizing”, the purpose of which is to improve surface properties (such as smoothness, permeability, printability, etc) of the substrate for subsequent use thereof in different processes, e.g., papers for printing and writing. This process consists of applying chemicals (e.g. starch among others) on the surface of the paper (size press, coating, etc) during the paper manufacturing process. With respect to the antioxidant and antimicrobial capacities, many foods are known to deteriorate and lose quality during transport, processing and storage, said foods being contaminated through microorganisms, chemical reactions and physical changes. Among these degradation models, microbial deterioration and oxidation reactions have the greatest impact. The antioxidant and antimicrobial property in paper is of interest from the viewpoint of food preservation, because paper with antioxidant or antimicrobial properties could be implemented in manufacturing a containment packaging with advanced properties (4).
The specific case of functionalizing individual cellulose fibers by means of biotechnological processes using enzymes has already been described, for example, for the hydrophobization of fibers (5), or the incorporation of natural phenols susceptible to conferring antioxidant properties or bacteriostatic properties, or to increasing mechanical strength (6). In fact, there are two patents where an oxidoreductase enzyme (laccase) is applied directly to the suspension of fibers, i.e., before the substrate is formed. One of these patents is for improving the wet strength of the paper (U.S. Pat. No. 6,610,172 B1), and the other patent is for the internal sizing of the paper (WO 11/009,979). These enzymatic treatments in the paper-making industry are designed for being applied on the fibrous suspension, in the mixing boxes and before forming the sheet. This entails several drawbacks from the industrial viewpoint, such as, for example: i) a limitation in the work conditions (pH, temperature, consistency, reaction time, etc) which must be suitable for optimal enzyme action, ii) high consumption of the transferred reagent, iii) possible interferences in bonds between fibers or with other products of the process, and iv) difficulty in recirculating the generated effluents. These drawbacks mean that said enzymatic treatments described up until now are industrially unfeasible.
A possible solution that would reduce transferred reagent consumption, would not affect the binding capacity between fibers and would eliminate the difficulty in recirculating the effluents would be to perform enzymatic treatments on the surface of the cellulosic substrate instead of on the fibrous suspension. However, performing an enzymatic treatment directly on the surface of a cellulosic substrate is unfeasible due to the conditions under which the enzymatic reaction must take place, such as long reaction times, for example. In fact, the literature does not describe any application of enzymatic systems for functionalization purposes that is applied directly on the surface of paper in the forming table, before drying, or once the sheet has already been formed. There are references about surface biotreatments in wood (7) and in finished fabrics (8). Nevertheless, in these treatments the presence of the substrate (wood or fabric) is necessary at the time of the enzymatic reaction. For a paper-making process, there are no references to surface biotreatments applied on the paper, i.e., on a previously formed sheet.
Therefore, for the purpose of overcoming the drawbacks indicated above, the authors of the present invention provide a new product, called post-enzymatic preparation, for the application thereof directly on the surface of the cellulosic substrate that has already been formed, providing the following advantages:                1.—This product is a simple and versatile formulation that is easy to apply on the surface of the cellulosic substrate.        2.—Different post-enzymatic preparations or products can be prepared for conferring different properties to the substrate by changing only natural or synthetic compound/compounds in the formulation.        3.—The conditions of the enzymatic process do not vary much according to the natural or synthetic compound.        4.—This formulation therefore varies according to the final property to be acquired in the substrate, but without needing to modify reaction conditions, depending only on whether or not the techniques for facilitating disaggregation/dispersion of the compound to be transferred are needed.        5.—It need not be prepared in-situ at the time of being applied, so it can be supplied as a formulated product.        6.—The enzymatic reaction in preparing the product to be applied takes place before being used on the substrate, allowing application thereof on the surface at different points of the paper machine, such as for example in the forming table, before the drying section, after the drying section and even in the finished product.        7.—The high temperature conditions in the paper machine do not affect/jeopardize the enzymatic reaction because said reaction takes place outside the point of application, and therefore outside the process of manufacturing the substrate.        8.—Since the product is not applied until the sheet is already formed, the enzymatic treatment does not affect the binding capacity of the fibers or the process itself for forming the sheet of paper.        9.—Since this post-enzymatic product can be applied on the surface, the amounts of natural or synthetic compound are reduced, and furthermore said compound stays more on the surface, therefore being more effective.        10.—The rheological properties of the post-enzymatic preparation allow it to be applied on the surface by means of different methods: size press, sprayers, metering bar, immersion-impregnation, etc.        11.—The use of this product in the mill does not involve an additional investment in machinery because systems already existing in the paper machine can be used, and if that is not the case, the investment is not disproportionate because only sprayers and a storage tank for the post-enzymatic preparation would be needed.        12.—The product can be prepared in concentrated form to subsequently be diluted right before use, thereby reducing transport and storage costs, the size of the facilities for preparing the product and the energy consumed for preparing it.        
The product of the present invention therefore allows providing the substrate with different properties according to the natural or synthetic compound used in the formulation of the product. This innovative product consists of a “post-enzymatic” preparation, i.e., it is a preparation resulting from an enzymatic reaction, the final rheological conditions and characteristics of which allow it to subsequently be applied on the surface by means of different metering system, without interfering in the process of manufacturing the substrate.
The authors of the present invention previously patented according to patent document ES2352495B1 a method in which internal sizing of paper was achieved by means of an enzyme-mediator system. As indicated in the examples of said patent, the process consisted of previously mixing the cellulose fibers (which will subsequently form the paper) with a compound (mediator), and subsequently adding a laccase-type enzyme so that the enzymatic reaction takes place for a specific time in the presence of the cellulose fibers, and thereby developing the property of internal sizing in the paper once it is formed. Therefore, the earlier patent did not describe the isolated product of the present invention, which is the result of the reaction of a compound, as defined in the present invention, and an oxidoreductase enzyme, without the presence of cellulose fibers. Furthermore, this described product which is obtained once the enzymatic reaction has ended is applied on the surface of the substrate, and furthermore provides different properties.
The present invention allows obtaining a product derived from the enzymatic reaction without the presence of cellulose fibers (an external agent in general) and outside the paper manufacturing process, being able to subsequently apply the product on the surface of the paper. Therefore, this product can be manufactured outside the paper-making industry as a chemical additive and subsequently be used by the paper-making company, using current technology and without jeopardizing the process of manufacturing the substrate.
Additionally, unlike the earlier patent (ES2352495B1), the product obtained in the present invention is a preparation (post-enzymatic product) derived from the enzymatic reaction; in contrast, the product obtained in ES2352495B1 consists of modified cellulose fibers in an aqueous suspension the production of which has always been within the process of manufacturing paper, entailing significant drawbacks making the industrial application thereof unfeasible. However, the present invention does not involve any drawback for being applied on an industrial level.
In addition, the earlier patent (ES2352495B1) mentions producing an internal sizing, whereas different properties (hydrophobicity, antioxidant and antimicrobial capacity, wet strength, among others) are achieved in the present invention.